The application of newly available genetic information to advances in preventative medicine and disease treatment requires efficient and accurate DNA detection technologies.1,2 One focus of recent technological developments is systems that exploit differential DNA hybridization at solid surfaces.3-6 In theory, hybridization of target sequences representing microbial genomic fragments or human disease-related genes to immobilized probe sequences would permit high-sensitivity and high-throughput DNA detection. Moreover, if closely related sequences could be discriminated, microbial pathogens could be detected and identified.
A variety of spectroscopic and analytical techniques can be used to detect DNA hybridization at surfaces.7-22 DNA-modified gold nanoparticles can be used to detect DNA sequences using optical and fluorescence spectroscopy.12,18 Surface plasmon resonance also provides a means to monitor hybridization of target sequences to DNA-modified gold substrates in real time.3,4,19,20,22 The results obtained with these methods indicate that high-sensitivity DNA detection can be achieved when immobilized oligonucleotides are used to capture sequences from solution.
Other gene detection methods (e.g., U.S. Pat. No. 5,972,692, and U.S. Pat. No. 5,312,527) do not use an electrocatalytic assay for DNA hybridization detection.
The detection of DNA sequences using electrochemical readout is particularly attractive for the development of clinical diagnostics.2,6,23,24 Quantitative electrochemical measurements of this type can be made using compact and inexpensive instrumentation, and covalently labeling DNA samples with reporter groups is typically unnecessary, simplifying sample preparation procedures. Indeed, a number of methods have been reported for the electrochemical detection of DNA, most of which rely on the signal produced by a noncovalently bound redox-active reporter group that is increased when DNA is hybridized to a surface modified with a probe sequence.7-11,13,15,21 In addition, single-base substitutions producing base mismatches within DNA duplexes immobilized on gold surfaces can be detected electrochemically using intercalating probes.14,16 The interruption of base stacking caused by the mismatch attenuates the current flowing to the reporter by interfering with DNA-mediated electronic coupling. This effect would potentially permit the electrochemical detection of disease-related point mutations.
Electrocatalytic processes that amplify the signals obtained at DNA-modified electrode surfaces provide a powerful means to increase the sensitivity and accuracy of a detection assay. Electrochemically-generated Ru(bpy)33+ reacts with guanines contained within a hybridized target in a catalytic process that generates large signals that can be used to detect DNA hybridization, albeit with limitations because of sequence dependence.13,15,21,24 In addition, an electrocatalytic reaction between an intercalating probe, methylene blue, and solution-borne Fe(CN)63− has been used to amplify the signal changes reporting the presence of mismatch-producing point mutations.16 However, neither system is ideal for hybridization-based detection of closely related sequences.